title: DNA-enzyme conjugate with a weak inhibitor that can specifically detect thrombin in a homogeneous medium.
abstract: We present the DNA-assisted control of enzymatic activity for the detection of a target protein using a new type of DNA-enzyme conjugate. The conjugate is composed of an enzyme inhibitor to regulate enzyme activity and a DNA aptamer to be responsive toward the analyte protein. Glutathione S-transferase (GST) and thrombin were selected as a model enzyme and an analyte protein. A hexahistidine tag was genetically attached to the C terminus of the GST, and the 5' end of an oligonucleotide was conjugated with nitrilotriacetic acid (NTA) for the site-specific conjugation of the DNA with the GST based on a Ni(2+) complex interaction. We found that fluorescein acted as a weak inhibitor of GST and succeeded in the regulation of GST activity by increasing the local concentration of the weak inhibitor by the hybridization of a 3'-end fluorescein-modified DNA. The catalytic activity of the DNA aptamer-enzyme conjugate showed a dose-dependent response to thrombin, indicating that the GST activity was clearly recovered by the binding of the DNA aptamer to thrombin. The current system enables the sensitive and specific detection of thrombin simply by measuring the enzymatic activity in a homogeneous medium.
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Fukuoka, Japan.
authors.forename: Josui
authors.initials: J
authors.lastname: Shimada
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Maruyama
authors.forename: Momoko
authors.initials: M
authors.lastname: Kitaoka
authors.forename: Noriho
authors.initials: N
authors.lastname: Kamiya
authors.forename: Masahiro
authors.initials: M
authors.lastname: Goto
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods*; D019793:Fluorescein / Q000378:metabolism; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D012680:Sensitivity and Specificity; D013917:Thrombin / Q000032:analysis* / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D005982:Glutathione Transferase; D013917:Thrombin; D019793:Fluorescein